Constructing a mammalian artificial chromosome (MAC) carrying the known functional elements required for chromosome replication, stable extra-chromosomal maintenance and segregation in a manipulatable form will be of great value not only for basic studies concerning the organization and function of mammalian chromosomes but also as a vector to introduce DNA segments (genes) of interest to test their functions in mammalian cells or bodies, since the genes carried by MACs will neither be subject to variable expression due to integration position effects nor cause unpredictable insertion mutation in the host chromosomes. Furthermore, a MAC will have the capacity to accommodate a DNA segment having a size in the megabase range, wherein an entire large gene or group of genes and regulatory elements could be included. For these reasons, MACs will offer exciting alternative vectors to currently existing vectors for somatic gene therapy, because frequently used infectious vectors derived from viruses are either integrated randomly into host chromosomes or exist extra-chromosomally but only transiently. Besides, these vectors are able to carry only short DNA segments. A new way to generate transgenic mice will be provided by the invention of MACs, if the stability of MACs during meiosis is established in mammalian development. However, the construction of a MAC has not yet been achieved due to technical difficulties (Willard, Proc. Natl. Acad. Sci. 93, 6874-6850,1996).
Yeast artificial chromosomes (YACs) has been constructed (Burke et al., Science, 236, 806-812,1987) with three essential DNA elements from the budding yeast, Saccharomyces cerevisiae; namely, an origin of replication or autonomously replicating sequence (ORI or ARS) is required for initiation of DNA replication, telomere sequences (TEL) are required to stabilize and facilitate complete replication of chromosomal ends, and a centromere (CEN) is required for faithful segregation of the sister chromatids after replication. As a result, YACs have become a major tool for cloning large gene segments of complex genomes. Similar to YACs, MACs are believed to be constructable with the three essential elements derived from mammalian genomes. Among the three, telomeres have been isolated from mammalian chromosomes and used for the mammalian chromosome manipulation (Brown et al., Hum. Mol. Genet., 27-1237,1994; Farr et al., EMBO J., 14, 5444-5454,1995), but centromeres and the origins of replication for mammalian chromosomes were found to be difficult to isolate because of the unavailability of activity assays.